Method of decreasing radiation or radio-mimetic chemotherapy for hematopoietic pluripotent cell engraftment

ABSTRACT

The present invention provides a method of engrafting donor mammalian hematopoietic pluripotent cells in a mammalian recipient using a decreased amount of radiation, comprising: (a) administering to the recipient at least one dosage of a hematopoietic growth factor; (b) subjecting the recipient to a low dosage of radiation; and (c) transplanting the donor hematopoietic pluripotent cells in the recipient, thereby engrafting the donor mammalian hematopoietic pluripotent cells in the mammalian recipient using a decreased amount of radiation. The invention also provides a method of engrafting donor mammalian hematopoietic pluripotent cells in a mammalian recipient using a decreased amount of radiomimetic compound, comprising: (a) administering to the recipient at least one dosage of a hematopoietic growth factor; (b) subjecting the recipient to a low dosage of radiomimetic compound; and (c) transplanting the donor hematopoietic pluripotent cells in the recipient, thereby engrafting the donor mammalian hematopoietic pluripotent cells in the mammalian recipient using a decreased amount of radiomimetic compound.

[0001] This is a divisional of, and claims priority to divisional patentapplication U.S. Ser. No. 09/468,727, filed Dec. 21, 1999, whichapplication claims priority to patent application U.S. Ser. No.08/983,532, filed Apr. 10, 1998, now U.S. Pat. No. 6,103,694, which is a35 U.S.C. §371 patent application of PCT/US96/12368, filed Jul. 22,1996, which status is abandoned, from provisional patent ApplicationU.S. Serial No. 60/001,386, filed Jul. 21, 1995, which applications arehereby incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the field of improving theengraftment of hematopoietic stem and progenitor cells in humanrecipients to treat disease. The invention also relates to a method fordecreasing radiation or chemotherapy for hematopoietic pluripotent cellengraftment.

[0004] 2. Background Art

[0005] The practice of bone marrow transplantation (BMT) or peripheralblood stem cell transplantation (PBSCT) involves placing a suspension ofdonor hematopoietic pluripotent cells (HPCs) into the blood stream ofthe recipient. HPC transplantations are currently performed with arecipient pre-conditioning regimen of high dosage radiation and/orchemotherapy. The goal of these treatments is to create an environmentin the recipient in which the donor's HPCs can successfully engraft byhoming into the recipient's bone marrow to further undergohematopoiesis. There may be several objectives for the use of suchpre-conditioning regimens, including eliminating cells underlyingdiseases such as leukemia, or lymphoma, or serving an immunosuppressivefunction to mitigate graft rejection in the treatment of non-cancerousdiseases. Conditioning regimens overall have the desirable effect oferadicating endogenous HPCs to make available more homing sites fortransplanted HPCs to successfully engraft. It is believed that currentradiation treatments allow homing and engraftment of transplanted stemcells by directly damaging or depleting the recipient's own stem cells,other hematopoietic regulatory cells, bone marrow stroma, and/or themicrocirculatory system. Tavassoli, M., “The role of conditioningregimens in homing of transplanted hemopoietic cell,” Bone MarrowTransplantation, 10: 15-17 (1992).

[0006] In clinical practice, radiation has been used primarily in highdoses to eliminate cells underlying cancerous diseases and toimmunosuppress graft rejection. Currently, patients are irradiated withapproximately 500 to 1600 cGy at single doses or in fractions. However,the use of radiation can have lethal toxic effects in the recipient, dueto the depletion of mature functional blood cells and damage to otherorgan systems. Therefore, non-myeloablative pre-transplantationregimens, which both minimize radiation effects and attain effectivelevels of engraftment, are very desirable.

SUMMARY OF THE INVENTION

[0007] The present invention provides a method of engrafting donormammalian hematopoietic pluripotent cells in a mammalian recipient usinga decreased amount of radiation, comprising: a. administering to therecipient at least one dosage of a hematopoietic growth factor; b.subjecting the recipient to a low dosage of radiation; and, c.transplanting the donor hematopoietic pluripotent cells into therecipient, thereby engrafting the donor mammalian hematopoieticpluripotent cells in the mammalian recipient using a decreased amount ofradiation.

[0008] The invention also provides a method of engrafting donormammalian hematopoietic pluripotent cells in a mammalian recipient usinga decreased amount of radiomimetic compound, comprising: a.administering to the recipient at least one dosage of a hematopoieticgrowth factor; b. subjecting the recipient to a low dosage ofradio-mimetic compound; and, c. transplanting the hematopoieticpluripotent cells into the recipient, thereby engrafting the donormammalian hematopoietic pluripotent cells in the mammalian recipientusing a decreased amount of radio-mimetic compound.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a bar graph showing the cytokine hematopoietic growthfactor pretreatment conditioning effect on engraftment of HPCs following200 cGy radiation in mice, one month after transplantation. RecipientC57BL/6J-Ly-5.1-Pep^(3b) mice received b.i.d. sc injections of PBS (□)0.1% BSA, 4 μg rhGCSF (□) in 0.1% BSA, or 4 μg rhGCSF plus 1.0 μgrmSCF(▪) in 0.1% BSA (R&D Systems, Minneapolis, Minn.) for four days. Onthe fifth day, mice received a single sc injection followed one hourlater by a 200 cGy radiation dose. Four hours after irradiation, allmice received 0.5×10⁶ Sca-1+BM cells via tail vein injection. Mice werebled 1 month after BMT and PB was analyzed for engraftment. Bothtreatment groups demonstrated at least a 3 fold increase in engraftmentcompared with controls (p<0.01). The data represent three experiments inseries with 2 mice per condition in each experiment. All animalssurvived this procedure.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention may be understood more readily by referenceto the following detailed description of specific embodiments includedherein. Although the present invention has been described with referenceto specific details of certain embodiments thereof, it is not intendedthat such details should be regarded as limitations upon the scope ofthe invention. The entire text of the references mentioned herein arehereby incorporated in their entireties by reference.

[0011] As used in the claims, “a” may mean one or more than one,depending upon the context within which it is used. The term“hematopoietic pluripotent cells” (HPCs) is used herein to describe bothundifferentiated stem cells or partially committed progenitor cellswhich would be transferred in either a bone marrow transplantation (BMT)or a peripheral blood stem cell transplantation (PBSCT). BMT techniquesare very well-established. Methods to obtain purified hematopoietic stemcells are also well-known. See for example: U.S. Pat. Nos. 5,061,620;5,087,570; 4,714,680; and 4,965,204.

[0012] The present invention provides a method of engrafting donormammalian hematopoietic pluripotent cells in a mammalian recipient usinga decreased amount of radiation, comprising: a. administering to therecipient at least one dosage of a hematopoietic growth factor; b.subjecting the recipient to a low dosage of radiation; and, c.transplanting the donor hematopoietic pluripotent cells into therecipient, thereby engrafting the donor mammalian hematopoieticpluripotent cells in the mammalian recipient using a decreased amount ofradiation. General cellular transplantation techniques are generallyfollowed with the use of the recipient preconditioning as describedherein. Preferably, the transplantation is autologous or allogenic.However, the transplantation can also be xenogenic. Such transplantationmay require the additional use of immunosuppressive compounds.

[0013] Hematopoietic growth factors are glycoprotein cytokines thatregulate the proliferation and differentiation of hematopoieticprogenitor cells. The hematopoietic growth factors intended to be usedin the present invention can be selected from the group GCSF(granulocyte colony stimulating factor), SCF (stem cell factor), GMCSF(granulocyte macrophage colony stimulating factor), IL-1(interleukin-1), IL-3, IL-6, IL-8, IL-11, IL-12, LIF (leukemiainhibitory factor), FGF-β (fibroblast growth factor β), FLT3, or acombination thereof. These growth factors can be purchased (e.g. R&DSystems, Minneapolis, Minn.) or made following procedures set forth inthe art generally and in publications describing the factors.Additionally, the hematopoietic growth factor can be a modified form ofthe factor or a fusion protein of hematopoietic growth factors selectedfrom the group GCSF, SCF, GMCSF, IL-1, IL-3, IL-6, IL-8, IL-11, IL-12,LIF, FGF-β, and FLT3. For example, PIXY321 is a fusion protein of GMSCFand IL-3. Modified growth factors (e.g. muteins) and fusion proteins canbe made according to methods known in the art. See, e.g. (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). Hematopoietic growthfactors, fusion, modifications, and combinations can be pre-screened forefficacy in the methods set forth in the Examples.

[0014] The hematopoietic growth factor can be administered to therecipient over a period ranging from about one day to two weeks prior toradiation or the administration of radio-mimetic compounds. Morepreferably, the hematopoietic growth factor can be administered to therecipient over about a five day period. The hematopoietic growth factoradministered is preferably from about 0.1 to 200 μg/kg/day for aboutfive days prior to subjecting the recipient to the low dosage ofradiation or radio-mimetic compound. Determining the exact dosage ofhematopoietic growth factor depends upon the type of cytokine beingadministered, and the condition of the patient, among other factorsknown to the skilled artisan. See, e.g., Remington's PharmaceuticalSciences, latest edition, by E. W. Martin Mack Pub. Co., Easton, Pa.

[0015] The step of subjecting the recipient to low dosage radiation canalso be performed within about the same day as the final hematopoieticgrowth factor administration. The low dosage radiation is preferablyfrom about 10 to 500 cGy, and more preferably, is about a 200 cGydosage.

[0016] The transplantation of hematopoietic pluripotent cells in mice ispreferably a transplantation of about 0.5×10⁶ SCA-1+cells. Thetransplantation of hematopoietic pluripotent cells in humans ispreferably a transplantation of about 10 to 500×10⁶ CD34+ cells.

[0017] The invention also provides a method of engrafting donormammalian hematopoietic pluripotent cells in a mammalian recipient usinga decreased amount of radiomimetic compound, comprising: a.administering to the recipient at least one dosage of a hematopoieticgrowth factor; b. subjecting the recipient to a low dosage ofradio-mimetic compound; and, c. transplanting the hematopoieticpluripotent cells into the recipient, thereby engrafting the donormammalian hematopoietic pluripotent cells in the mammalian recipientusing a decreased amount of radio-mimetic compound. In this method, thepresently preferred radio-mimetic compounds are busulfan or BCNU, or acombination thereof. The radio-mimetic compound can be administered at adosage ranging from LD_(0.1) to LD₅₀. See e.g. Down et al. “Transientand permanent engraftment potential of murine hematopoietic stem cellsubsets: differential effects of host conditioning with gamma radiationand cytotoxic drugs,” Exp. Hem. 21:913-921 (1993).

[0018] Allogenic and autologous HPC transplantation currently utilizesrecipient preconditioning consisting of ablative radiation and/orchemotherapy to ensure successful engraftment of donor stem cells.Typically, transplantations are performed as a rescue strategy followinghigh-dose ablation for cancer therapy in the autologous setting, or asan engraftment strategy following high-dose ablation for immunologicalsuppression in the allogenic setting. At the doses required, thesecurrent therapies have significant multi-organ toxic effects.

[0019] Many diseases treatable with HPC transplantation do not requirehigh dose ablation, however high dose ablation has previously beenrequired to obtain suitable HPC engraftment. The present discoverypermits improved HPC engraftment in patients without the need toadminister life threatening levels of ablative therapy. This inventioncan greatly improve the survival and cure rates of numeroushematopoietic diseases which do not require high dose ablation whichcurrently rely on the transplantation of HPCs. For example, diseases andconditions which can be treated by the methods of the present inventioninclude: congenital B- and T-lymphocyte disorders, such as predominantlyantibody defects, X-linked agammaglobulinemia, common variableimmunodeficiency, immunodeficiency with thymoma, selective IgAdeficiency, X-linked immunodeficiency with hyper-IgM, antibodydeficiency with normal immunoglobulins, subclass deficiency, poorresponse to polysaccharide antigens, or X-linked lymphoproliferativesyndrome; or a combined immunodeficiency-primary defect in cellularimmunity, such as severe combined immunodeficiency, autosomal recessiveand X-linked, adenosine deaminase deficiency, defective expression ofhistocompatibility antigens, deficiency of T-cell receptors, Omen'ssyndrome, cellular immunodeficiency with immunoglobulins (Nezelof'ssyndrome), purine nucleoside phosphorylase deficiency; or an immunedeficiency associated with other defects, such as Wiskoff-Aldrichsyndrome, ataxia telangiectasia, cartilage-hair hypoplasia,hyperimmunoglobulin E syndrome, or chronic mucocutaneous candidiasis.The invention may treat disorders of phagocytic function, such asdisorders of production and consumption, abnormal production, Kostmann'ssyndrome, Schwachman's syndrome, cyclic neutropenia, Primary B- andT-lymphocyte disorders, X-linked hyper-IgM, X-linked agammaglobulinemia,ataxia telangiectasia, cartilage-hair hypoplasia, IgA deficiency;disorders of migration and chemotaxis, general defects in leukocytemobility, nonspecific disorders, such as Kartogener's syndrome, lazyleukocyte syndrome, hyper-IgE syndrome, Chédiak-Higashi syndrome; ordisorders of intracellular killing, such as chronic granulomatousdisease, myeloperoxidase deficiency, gluthathione reductase andperoxidase deficiency, glucose-o-phosphate dehydrogenase deficiency; ora deficiency of leukocyte function antigen 1 (LFA-1).

[0020] The invention can also be useful for indications for bone marrowtransplantation, such as hematologic disorders, marrow aplasia,Fanconi's aplasia, Diamond-Blackfan syndrome, hemoglobinopathies,β-thalassemia major, sickle cell anemia, neutrophil disorders,congenital neutropenia, chronic granulomatous disease, Chédiak-Higashisyndrome, Osteopetrosis; immune deficiency disorders, such as severecombined immunodeficiency disease, ADA-deficient SCID, reticulardysgenesis, bare lymphocyte syndrome, PNP deficiency, LFA-1 deficiency,ataxia telangiectasia, or Wiskoff-Aldrich syndrome; metabolic disorders,such as mucopolysaccharidoses, Hurler's syndrome, Hunter's syndrome,Sanfilippo's syndrome, leukodystrophies, metachromatic leukodystrophy,adrenoleucodystrophy, sphingolipidoses, Neimann-Pick syndrome, Gaucher'sdisease; or in the setting of HIV infection, red cell membrane disorders(such as hereditary spherocytosis, etc.), G-6-PD deficiency, paroxysmalnocturnal hemoglobinuria, myelodysplastic syndrome, or aplastic anemia,for example. Furthermore, the discovery has useful implications forimproving the efficacy of gene therapies using HPCs, especially in thosecontexts where less than 100% HPC replacement is required foreffectiveness.

[0021] The growth factors used in the invention can be convenientlyformulated into pharmaceutical compositions composed of one or more ofthe compounds in association with a pharmaceutically acceptable carrier.See, e.g., Remington's Pharmaceutical Sciences, referenced above, whichdiscloses typical carriers and conventional methods of preparingpharmaceutical compositions that may be used in conjunction with thepreparation of formulations of the inventive compounds and which isincorporated by reference herein. By “pharmaceutically acceptable” ismeant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to an individual along with thegrowth factor without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical composition in which it is contained.

[0022] The growth factors may be administered orally, parenterally(e.g., intravenously), by intramuscular injection, by intraperitonealinjection, topically, transdermally, or the like, although subcutaneousinjection is preferred. The amount of active compound administered will,of course, be dependent on the subject being treated, the subject'sweight, the manner of administration and the judgment of the prescribingphysician. Generally, however, administration and dosage willapproximate that which is typical for the administration of naturallyoccurring proteins, especially hematopoietic growth factors. The optimaldosages of the particular hematopoietic growth factors may routinely bedetermined by a skilled artisan using currently available techniques andreferences.

[0023] Depending on the intended mode of administration, thepharmaceutical compositions may be in the form of solid, semi-solid orliquid dosage forms, such as, for example, tablets, suppositories,pills, capsules, powders, liquids, suspensions, lotions, creams, gels,or the like, preferably in unit dosage form suitable for singleadministration of a precise dosage. The compositions will include, asnoted above, an effective amount of the selected hematopoietic growthfactors in combination with a pharmaceutically acceptable carrier and,in addition, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, diluents, etc.

[0024] Liquid pharmaceutically administrable compositions can, forexample, be prepared by dissolving, dispersing, etc., an active compoundas described herein and optional pharmaceutical adjuvants in anexcipient, such as, for example, water, saline aqueous dextrose,glycerol, ethanol, and the like, to thereby form a solution orsuspension. For solid compositions, conventional nontoxic solid carriersinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose,sucrose, magnesium carbonate, and the like. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example seeRemington's Pharmaceutical Sciences, referenced above.

[0025] Parental administration is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parental administration involves use of aslow release or sustained release system, such that a constant level ofdosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

EXAMPLES

[0026] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compounds claimed herein are made and evaluated, and areintended to be purely exemplary of the invention and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to ensure accuracy with respect to numbers (e.g.,amounts, etc.) but some errors and deviations should be accounted for.

[0027] The data which forms the basis of the present invention concernsthe enhanced in vivo transfer of purified SCA-1+bone marrow derived HPCsusing congenic C57BL/6 mice, which differ only at the Ly5 locus. Theorigin of a cell is detectable by immunoreactivity with eitheranti-Ly5.1 or anti-Ly5.2 antibodies by FACS of PBMCs. See, e.g. Flemminget al., “Functional Heterogeneity is associated with the cell cyclestatus of murine hematopoietic stem cells,” J. Cell Biol. 122(4):897-902(1993); Wineman et al., “CD4 is expressed on murine pluripotenthematopoietic stem cells,” Blood 80(7):1717-1724 (1992); Wineman et al.,“Maintenance of high levels of pluripotent hematopoietic stem cells invitro: effect of stromal cells and c-kit,” Amer. Soc. Hem. (1993). Usingthis system, the effects of pre-transplantation cytokine conditioning onshort term engraftment, defined as the percentage of total PBMCs in therecipient that were donor Ly5.2 cells, measured one month aftertransplantation, have been determined.

[0028] Recipient C57BL/6-Ly-5.1-Pep^(3b) mice received twice dailysubcutaneous injections of either vehicle control (0.1% BSA), 4 μg(about 200 μg/kg) recombinant human granulocyte colony stimulatingfactor (rhGCSF) (R&D Systems Minneapolis, Minn.) in 0.1% BSA, or 4 μgrhGCSF+1.0 μg (about 50 μg/kg) recombinant murine stem cell factor(rmSCF) (R&D Systems Minneapolis, Minn.) in 0.1% BSA, for four days. Onthe fifth day, all mice received a final injection of hematopoieticgrowth factor or control, and within the about one hour a 200 cGyirradiation dose followed about four hours later by transplantation of0.5×10⁶ donor Sca-1+HPC via tail vein injection. Cytokine treatmentceased after transplantation. After one month, mice were bled and thosereceiving rhGCSF or rhGCSF+rmSCF showed at least a three fold increasein engraftment compared with those receiving control (18.9% and 20.6%versus 5.6%), as shown in FIG. 1. All animals remained healthy duringthe 1 month of observation reported herein.

What is claimed is:
 53. A method of engrafting donor mammalianhematopoietic pluripotent cells in a mammalian recipient using adecreased amount of radiomimetic compound, comprising: a. administeringto the recipient at least one dosage of a hematopoietic growth factorselected from the group consisting of GCSF, SCF, GMCSF, IL-1, IL-3,IL-6, IL-8, IL-11, IL-12, LIF, FGF-*, FLT3, and PIXY321, or acombination thereof; b. subjecting the recipient to a low dosage ofradio-mimetic compound; and, c. transplanting the hematopoieticpluripotent cells into the recipient, thereby engrafting the donormammalian hematopoietic pluripotent cells in the mammalian recipientusing a decreased amount of radio-mimetic compound.
 54. A method ofengrafting donor mammalian hematopoietic pluripotent cells in amammalian recipient using a decreased amount of radiomimetic compound,comprising: a. administering to the recipient at least one dosage of ahematopoietic growth factor fusion protein comprising hematopoieticgrowth factors selected from the group consisting of GCSF, SCF, GMCSF,IL-1, IL-3, IL-6, IL-8, IL-11, IL-12, LIF, FGF-*, and FLT3; b.subjecting the recipient to a low dosage of radio-mimetic compound; and,c. transplanting the hematopoietic pluripotent cells into the recipient,thereby engrafting the donor mammalian hematopoietic pluripotent cellsin the mammalian recipient using a decreased amount of radio-mimeticcompound.
 55. A method of engrafting autologous mammalian hematopoieticpluripotent cells in a mammalian patient using a decreased amount ofradio-mimetic compound, comprising: a. administering to the patient atleast one dosage of a hematopoietic growth factor selected from thegroup consisting of GCSF, SCF, GMCSF, IL-1, IL-3, IL-6, IL-8, IL-11,IL-12, LIF, FGF-*, FLT3, and PIXY321, or a combination thereof; b.subjecting the patient to a low dosage of radio-mimetic compound; and,c. transplanting the hematopoietic pluripotent cells into the patient,thereby engrafting the autologous mammalian hematopoietic pluripotentcells in the patient using a decreased amount of radio-mimetic compound.56. A method of engrafting autologous mammalian hematopoieticpluripotent cells in a mammalian patient using a decreased amount ofradio-mimetic compound, comprising: a. administering to the patient atleast one dosage of a hematopoietic growth factor fusion proteincomprising hematopoietic growth factors selected from the groupconsisting of GCSF, SCF, GMCSF, IL-1, IL-3, IL-6, IL-8, IL-11, IL-12,LIF, FGF-*, and FLT3; b. subjecting the patient to a low dosage ofradio-mimetic compound; and, c. transplanting the hematopoieticpluripotent cells into the patient, thereby engrafting the autologousmammalian hematopoietic pluripotent cells in the patient using adecreased amount of radio-mimetic compound.